High-speed on demand microfluidic droplet generation and manipulation

ABSTRACT

Methods and devices for the formation and/or merging of droplets in microfluidic systems are provided. In certain embodiments a microfluidic droplet merger component is provided that comprises a central channel comprising a plurality of elements disposed and spaced to create a plurality of lateral passages that drain a carrier fluid out of a fluid stream comprising droplets of a first fluid contained in the carrier fluid; and a deformable lateral membrane valve disposed to control the width of said center channel.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of and priority to U.S. Ser. No.61/798,516, filed on Mar. 15, 2013, which is incorporated herein byreference in its entirety for all purposes.

STATEMENT OF GOVERNMENTAL SUPPORT

This invention was made with Government support under Grant No. ECCS0901154, awarded by the National Science Foundation. The Government hascertain rights in this invention.

BACKGROUND

Over the last decade microfluidic systems have developed into valuableinstrumental platforms for performing high throughput chemistry andbiology (deMello (2006) Nature, 442: 394-402). The ability tocontrollably merge droplets within segmented flow systems is of highimportance when performing complex chemical or biological analyses(Shestopalov et al. (2004) Lab Chip, 4: 316-321). Unfortunately, thecontrolled merging of multiple droplets in a sequential fashion is notstraightforward. Although the emulsions produced in microfluidic systemsare thermodynamically metastable, the process of merging has not provento be predictable, due to subtle variations in interfacial tension,surface topography of microchannels, and fluidic properties such as ofdroplet size, viscosity, and velocity (see, e.g., Fuerstman et al.(2007) Science, 315: 828-832).

Droplet merging is important or essential in many applications includingsequential reactions (Kim et al. (2006) Anal. Chem., 78(23): 8011-8019),multiple step manipulation of cells (He et al. (2005) Anal. Chem.,77(6): 1539-1544), high-throughput bioassays (Srisa-Art et al. (2007)Anal. Chem., 79: 6682-6689), and the like. Additionally, the ability tomerge and split droplets or bubbles in a high throughput manner cabimpact the use of bubble logic systems for exchanging chemical andelectronic information (Prakash and Gershenfeld (2007) Science,315(5813): 832-835).

In typical droplet merging processes, relatively large time and spatialscales are involved. For example, timescales may range from thesub-microsecond regime for some chemical reactions to many hours andeven days for cell-based assays. Similarly, large spatial scales alsoexist, for example, between the droplets to be merged and between thedroplets and the component interfaces that interact to drive the mergingprocess.

Several techniques have been developed to merge droplets. These areeither active and involve components such as electric fields (Priest etal. (2006) Appl. Phys. Lett., 89: 134101:1-134101:3; Ahn et al. (2006)Appl. Phys. Lett., 88: 264105), or passive and utilize the surfaceproperties (Fidalgo et al. (2007) Lab Chip, 7(8): 984-986) or structure(Tan et al. (2004) Lab Chip, 4(4): 292-298) of the fluidic conduit.

SUMMARY

In various embodiments in various embodiments, a microfluidic dropletmerger component (e.g., for integration into microfluidic systems suchas lab-on-a-chip systems, and the like) is provided. An illustrative,but noon-limiting droplet merger structure comprises a central channelcomprising a plurality of elements (e.g. a micropillar array) disposedand spaced to create a plurality of lateral passages that drain acarrier fluid out of a fluid stream comprising droplets of a first fluidcontained in said carrier fluid; and a deformable lateral membrane valvedisposed to control the width of said center channel. Trapping andmerging of different numbers of droplets can be controlled by thespacing and arrangement of lateral passages and/or elements forming suchpassages (e.g., the micropillar array structure), the timing, and theconstriction size of the deformable lateral membrane valve. Thedeformable membrane (membrane valve) forms a controllable,variable-sized constriction, e.g., at the downstream of the trappingstructure. By controlling the constriction size and timing, differentnumbers of droplets can be trapped and merged before exiting the device.Also provided are microfluid droplet generators and devices comprisingone or more microfluid droplet generators and/or one or more dropletmerger structures.

In various aspects, the invention(s) contemplated herein may include,but need not be limited to, any one or more of the followingembodiments:

In various aspects, the invention(s) contemplated herein may include,but need not be limited to, any one or more of the followingembodiments:

Embodiment 1

A microfluidic droplet merger component, said component including: acentral channel including a plurality of elements disposed and spaced tocreate a plurality of lateral passages that drain a carrier fluid out ofa fluid stream including droplets of a first fluid contained in saidcarrier fluid; and a deformable lateral membrane valve disposed tocontrol the width of said center channel.

Embodiment 2

The droplet merger component of embodiment 1, wherein said membranevalve is a pneumatically actuated lateral membrane valve.

Embodiment 3

The droplet merger component according to any one of embodiments 1-2,wherein the width of said central channel reduces as a function ofdistance downstream through said plurality of lateral passages.

Embodiment 4

The droplet merger component according to any one of embodiments 1-3,wherein the where the width of said lateral passages is smaller than thewidth of said central channel at the same location and smaller than theaverage diameter of a droplet in the central channel.

Embodiment 5

The droplet merger component according to any one of embodiments 1-4,wherein said plurality of elements comprise a micropillar array.

Embodiment 6

The droplet merger component according to any one of embodiments 1-5,wherein said valve is located at or downstream of the last of saidplurality of elements.

Embodiment 7

The droplet merger component of embodiment 5, wherein said micropillararray includes pairs of pillars that form lateral channels slanted in adownstream direction.

Embodiment 8

The droplet merger component of embodiment 7, wherein said valve islocated at or downstream of the last (downstream) pairs of pillars.

Embodiment 9

The droplet merger component according to any one of embodiments 1-8,wherein said pillars are configured to provide an inter-pillar spacingthat ranges from about 0.1 μm to about 100 μm.

Embodiment 10

The droplet merger component according to any one of embodiments 1-8,wherein said pillars are configured to provide an inter-pillar spacingthat ranges from about 0.1 μm to about 10 μm.

Embodiment 11

The droplet merger component according to any one of embodiments 1-10,wherein said deformable lateral membrane valve is configured to form acontrollable, variable-sized construction at the downstream end of saidplurality of elements.

Embodiment 12

The droplet merger component according to any one of embodiments 1-11,wherein said deformable lateral membrane valve is configured to deformhorizontally.

Embodiment 13

The droplet merger component according to any one of embodiments 1-11,wherein said deformable lateral membrane valve is configured to deformvertically.

Embodiment 14

The droplet merger component according to any one of embodiments 1-13,wherein said micropillar array is formed from a material selected fromthe group consisting of glass, metal, ceramic, mineral, plastic, andpolymer.

Embodiment 15

The droplet merger component according to any one of embodiments 1-13,wherein said micropillar array is formed from an elastomeric material.

Embodiment 16

The droplet merger component of embodiment 15, wherein said elastomericmaterial is selected from the group consisting of polydimethylsiloxane(PDMS), polyolefin plastomers (POPs), perfluoropolyethylene (a-PFPE),polyurethane, polyimides, and cross-linked NOVOLAC® (phenol formaldehydepolymer) resin.

Embodiment 17

A microfluidic droplet generator, said generator including: a firstmicrofluidic channel containing a first fluid adjacent to a secondmicrofluidic channel containing a second fluid wherein said first fluidis substantially immiscible in second fluid; and a cavitation channel orchamber where the contents of said cavitation channel or chamber isseparated from the contents of said first microfluidic channel by adeformable channel wall or chamber wall, where said cavitation channelor chamber is configured to permit said deformable channel wall orchamber wall to deform when a bubble is formed in said cavitationchannel or chamber, and where said cavitation channel or chamber isdisposed above or below said first microfluidic channel.

Embodiment 18

The droplet generator of embodiment 17, wherein, said first microfluidicchannel is in fluid communication with said second microfluidic channelvia a port or a channel.

Embodiment 19

The droplet generator according to any one of embodiments 17-18, where afirst portion of said first microfluidic channel is disposed a firstdistance away from said second microfluidic channel, and a secondportion of said first microfluidic channel is disposed a second distanceaway from said second microfluidic channel and said second distance isless than said first distance.

Embodiment 20

The droplet generator of embodiment 19, wherein said first microfluidicchannel includes a third portion disposed so that said second portion islocated between said first portion and said third portion and said thirdportion of said microfluidic channel is located at a third distance awayfrom said second microfluidic channel and said third distance is greaterthan said second distance.

Embodiment 21

The droplet generator according to any one of embodiments 17-20, wherethe maximum width of said first microfluidic channel and/or said secondmicrofluidic channel ranges from about 0.1 μm to about 500 μm.

Embodiment 22

The droplet generator according to any one of embodiments 17-20, wherethe maximum width of said first microfluidic channel and/or said secondmicrofluidic channel ranges from about 50 μm to about 100 μm.

Embodiment 23

The droplet generator according to any one of embodiments 17-20, wherethe width of said first microfluidic channel and/or said secondmicrofluidic channel is about 100 μm.

Embodiment 24

The droplet generator according to any one of embodiments 17-23, wherethe maximum depth of said first microfluidic channel and/or said secondmicrofluidic channel ranges from about 0.1 μm to about 500 μm.

Embodiment 25

The droplet generator according to any one of embodiments 17-23, wherethe maximum depth of said first microfluidic channel and/or said secondmicrofluidic channel ranges from about 40 μm to about 80 μm.

Embodiment 26

The droplet generator according to any one of embodiments 17-23, wherethe typical depth of said first microfluidic channel and/or said secondmicrofluidic channel is about 50 μm.

Embodiment 27

The droplet generator according to any one of embodiments 17-26, whereinthe typical depth of said cavitation channel or chamber ranges fromabout 100 μm to about 150 μm.

Embodiment 28

The droplet generator according to any one of embodiments 17-27, whereinsaid droplet generator is configured to generate droplets having avolume ranging from about 1 atto L to about 1 μL.

Embodiment 29

The droplet generator of embodiment 28, wherein said droplet generatoris configured to generate droplets having a volume ranging from about 1μL to about 150 μL.

Embodiment 30

The droplet generator according to any one of embodiments 17-28, whereinsaid cavitation channel or chamber is a cavitation channel.

Embodiment 31

The droplet generator of embodiment 30, wherein said cavitation channelprovides permits the contents of said channel to flow and thereby aiddissipation of a bubble formed therein.

Embodiment 32

The droplet generator according to any one of embodiments 17-31, whereinsaid cavitation channel or chamber is disposed above said firstmicrofluidic channel.

Embodiment 33

The droplet generator according to any one of embodiments 17-31, whereinsaid cavitation channel or chamber is disposed below said firstmicrofluidic channel.

Embodiment 34

The droplet generator according to any one of embodiments 17-33, whereinsaid cavitation channel or chamber contains a dye.

Embodiment 35

The droplet generator according to any one of embodiments 17-33, whereinsaid cavitation channel or chamber contains light-absorbing nanoparticleand/or microparticles.

Embodiment 36

The droplet generator according to any one of embodiments 17-35, whereinsaid first microfluidic channel is configured to provide said firstfluid under a substantially static pressure to create a stable interfacebetween said first fluid and said second fluid.

Embodiment 37

The droplet generator according to any one of embodiments 17-36, whereinsaid first fluid includes an aqueous fluid.

Embodiment 38

The droplet generator according to any one of embodiments 17-37, whereinsaid second fluid includes an oil or an organic solvent.

Embodiment 39

The droplet generator of embodiment 38, wherein said second fluidincludes a solvent selected from the group consisting of carbontetrachloride, chloroform, cyclohexane, 1,2-dichloroethane,dichloromethane, diethyl ether, dimethyl formamide, ethyl acetate,heptane, hexane, methyl-tert-butyl ether, pentane, toluene, and2,2,4-trimethylpentane.

Embodiment 40

The droplet generator of embodiment 38, wherein said second fluidincludes an oil.

Embodiment 41

The droplet generator according to any one of embodiments 17-40, whereinsaid port or channel includes a nozzle.

Embodiment 42

The droplet generator according to any one of embodiments 17-41, whereinsaid first and/or second microfluidic channel is formed from a materialselected from the group consisting of glass, metal, ceramic, mineral,plastic, and polymer.

Embodiment 43

The droplet generator according to any one of embodiments 17-42, whereinsaid first and/or second microfluidic channel is formed from anelastomeric material.

Embodiment 44

The droplet generator of embodiment 43, wherein said elastomericmaterial is selected from the group consisting of polydimethylsiloxane(PDMS), polyolefin plastomers (POPs), perfluoropolyethylene (a-PFPE),polyurethane, polyimides, and cross-linked NOVOLAC® (phenol formaldehydepolymer) resin.

Embodiment 45

The droplet generator according to any one of embodiments 17-44, whereinsaid generator can provide on-demand droplet generation at a speed ofgreater than about 1,000, more preferably greater than about 2,000droplets/sec, more preferably greater than about 4,000 droplets/sec,more preferably greater than about 6,000 droplets/sec, or morepreferably greater than about 8,000 droplets/sec.

Embodiment 46

The droplet generator according to any one of embodiments 17-44, whereinsaid device can provide on-demand droplet generation at a speed rangingfrom zero droplets/sec, 1 droplets/sec, 2 droplets/sec, about 5droplets/sec, about 10 droplets/sec, about 20 droplets/sec, about 50droplets/sec, about 100 droplets/sec, about 500 droplets/sec, or about1000 droplets/sec, up to about 1,500 droplets/sec, about 2,000droplets/sec, about 4,000 droplets/sec, about 6,000 droplets/sec, about8,000 droplets/sec, about 10,000 droplets/sec, about 20,000droplets/sec, about 50,000 droplets/sec, or about 100,000 droplets/sec.

Embodiment 47

The droplet generator according to any one of embodiments 17-44, whereinsaid device can provide on-demand droplet generation at a speed ofgreater than about 1,000, more preferably greater than about 10,000,more preferably greater than about 20,000 droplets/sec, more preferablygreater than about 40,000, more preferably greater than about 50,000droplets/sec, more preferably greater than about 80,000, or morepreferably greater than about 100,000 droplets/sec.

Embodiment 48

The droplet generator according to any one of embodiments 17-47, whereinsaid generator is present in a system including an energy sourceconfigured to form a bubble in said cavitation channel or chamber.

Embodiment 49

The droplet generator of embodiment 48, wherein said energy sourceincludes an optical energy source or microwave emitter.

Embodiment 50

The droplet generator of embodiment 48, wherein said energy sourceincludes a laser.

Embodiment 51

The droplet generator of embodiment 50, wherein said energy sourceincludes a pulse laser.

Embodiment 52

The droplet generator according to any one of embodiments 17-51, whereinsaid generator is disposed on a substrate including a material selectedfrom the group consisting of a polymer, a plastic, a glass, quartz, adielectric material, a semiconductor, silicon, germanium, ceramic, and ametal or metal alloy.

Embodiment 53

The droplet generator according to any one of embodiments 17-52, whereinsaid generator is integrated with other microfluidic components.

Embodiment 54

The droplet generator of embodiment 53, wherein said other microfluidiccomponents selected from the group consisting of PDMS channels, wells,valves.

Embodiment 55

The droplet generator of embodiment 53, wherein said generator is acomponent of a lab-on-a-chip.

Embodiment 56

The droplet generator according to any one of embodiments 17-55, whereinsaid first fluid includes one or more reagents for polymerase chainreaction (PCR).

Embodiment 57

The droplet generator of embodiment 56, wherein said first fluidincludes one or more reagents selected from the group consisting of aPCR primer, a PCR template, a polymerase, and a PCR reaction buffer.

Embodiment 58

A device for the manipulation of microfluidic droplets, said deviceincluding a substrate carrying or including: one or more droplet mergercomponents according to any one of embodiments 1-16; and optionally oneor more droplet generators according to any one of embodiments 17-57.

Embodiment 59

The device of embodiment 58, wherein said device further includes acontroller that controls the amount and timing of constriction of saidmembrane valve.

Embodiment 60

The device according to any one of embodiments 58-59, wherein saiddevice includes one or more droplet generators according to any one ofembodiments 17-57.

Embodiment 61

The device according to any one of embodiments 58-60, wherein saiddevice includes at least two droplet generators.

Embodiment 62

The device of embodiment 61, wherein said device includes at least fourdroplet generators.

Embodiment 63

The device according to any one of embodiments 61-62, wherein aplurality of droplet generators are configured to share a common secondmicrofluidic channel and to inject droplets into said common secondmicrofluidic channel.

Embodiment 64

The device of embodiment 63, wherein a droplet merger component isdisposed to receive and merge droplets from said common secondmicrofluidic channel.

Embodiment 65

A system for the generation of droplets and/or the encapsulation ofparticles or cells said, said system including a droplet generatoraccording to any one of embodiments 17-57 and an excitation source forforming gas bubbles in a fluid.

Embodiment 66

The system of embodiment 65, wherein said excitation source includes anoptical energy source.

Embodiment 67

The system of embodiment 66, wherein said excitation source includes anon-coherent optical energy source.

Embodiment 68

The system of embodiment 66, wherein said excitation source includes alaser.

Embodiment 69

The system according to any one of embodiments 66-68, wherein saidsystem includes an objective lens configured to focus optical energyinto said cavitation channel or chamber.

Embodiment 70

The system of embodiment 69, wherein said system includes a half-waveplate.

Embodiment 71

The system according to any one of embodiments 69-70, wherein saidsystem includes a polarizer.

Embodiment 72

The system of embodiment 71, wherein said polarizer includes apolarizing beam splitter cube.

Embodiment 73

The system according to any one of embodiments 66-72, wherein saidsystem includes a controller that adjusts at least one of the timing ofoccurrence of light pulses emitted by the optical energy source, thefrequency of occurrence of pulses emitted by the optical energy source,the wavelength of pulses emitted by the optical energy source, theenergy of pulses emitted by the optical energy source, and the aiming orlocation of pulses emitted by the optical energy source.

Embodiment 74

The system according to any one of embodiments 65-73, wherein saidsystem further includes components for detecting particles, droplets, orcells in said system.

Embodiment 75

The system of embodiment 74, wherein said components comprise an opticaldetection system, an electrical detection system, a magnetic detectionsystem or an acoustic wave detection system.

Embodiment 76

The system of embodiment 74, wherein said components comprise an opticaldetection system for detecting a scattering, a fluorescence, or a ramenspectroscopy signal.

Embodiment 77

A method of combining droplets in a microfluidic system, said methodincluding providing a plurality of droplets flowing through amicrofluidic channel into the central channel(s) of one or more dropletmerger components according to any one of embodiments 1-16 causing themerger of a plurality of droplets.

Embodiment 78

The method of embodiment 77, further including varying the constrictioncreated by said lateral membrane valve to control the timing of dropletmerger and/or the number of merged droplets.

Embodiment 79

The method of embodiment 78, wherein said varying the constrictionincludes operating a controller that pneumatically actuates said lateralmembrane valve(s).

Embodiment 80

A method for generating droplets said method including: applying anenergy source to a droplet generator according to any one of embodiments17-57, where said energy source forms bubbles in said cavitation channelor chamber to deform said deformable channel wall or chamber wall and toinject a droplet of said first fluid into said second fluid in saidsecond microfluidic channel.

Embodiment 81

The method of embodiment 80, wherein said utilizing an energy sourceincludes utilizing a laser to excite cavitation bubbles in saidcavitation channel or chamber.

Embodiment 82

The method of embodiment 81, wherein said method includes using acontroller that adjusts at least one of the timing of occurrence ofpulses emitted by a pulsed laser, the frequency of occurrence of pulsesemitted by the pulsed laser, the wavelength of pulses emitted by thepulse laser, the energy of pulses emitted by the pulse laser, and theaiming or location of pulses emitted by the pulse laser.

Embodiment 83

The method according to any one of embodiments 80-82, further includinggenerating a plurality of separate and additional cavitation bubbles ata frequency of at least 1000 Hz.

Embodiment 84

The method of embodiment 83, wherein said method is repeated at afrequency of 1 kHz or greater.

Embodiment 85

The method according to any one of embodiments 80-84, wherein said firstfluid includes one or more reagents for polymerase chain reaction (PCR).

Embodiment 86

The droplet generator of embodiment 85, wherein said first fluidincludes one or more reagents selected from the group consisting of aPCR primer, a PCR template, a polymerase, and a PCR reaction buffer.

Embodiment 87

A method of generating and combining droplets, said method including:providing a device including one or more droplet generators dropletgenerator according to any one of embodiments 17-57 and one or moredroplet merger component(s) according to any one of embodiments 1-16,wherein at least one of said one or more droplet merger component(s) isdisposed to receive droplets generated by at least one of said one ormore droplet generators; applying an energy source a cavitation channelor chamber of one or more of said one or more droplet generator(s),where said energy source forms bubbles in said cavitation channel orchamber to deform said deformable channel wall or chamber wall and toinject a droplet of said first fluid into said second fluid in saidsecond microfluidic channel; receiving a plurality of droplets generatedby said one or more droplet generator(s) in at least one of said one ormore droplet merger components where said droplets merger to form acombined droplet fluid.

Embodiment 88

The method of embodiment 87, wherein said device includes a plurality ofdroplet generators.

Embodiment 89

The method of embodiment 87, wherein said device includes at least threedroplet generators.

Embodiment 90

The method according to any one of embodiments 87-89, wherein saiddevice includes a plurality of droplet merger components.

Embodiment 91

The method of embodiment 90, wherein said device includes at least threedroplet merger components.

Embodiment 92

The method according to any one of embodiments 87-91, wherein saidutilizing an energy source includes utilizing a laser to excitecavitation bubbles in said cavitation channel or chamber.

Embodiment 93

The method of embodiment 92, wherein said method includes using acontroller that adjusts at least one of the timing of occurrence ofpulses emitted by a pulsed laser, the frequency of occurrence of pulsesemitted by the pulsed laser, the wavelength of pulses emitted by thepulse laser, the energy of pulses emitted by the pulse laser, and theaiming or location of pulses emitted by the pulse laser.

Embodiment 94

The method according to any one of embodiments 87-93, further includinggenerating a plurality of separate and additional cavitation bubbles ata frequency of at least 1000 Hz.

Embodiment 95

The method of embodiment 94, wherein said method is repeated at afrequency of 1.2 kHz or greater.

Embodiment 96

The method according to any one of embodiments 87-95, wherein said firstfluid includes one or more reagents for polymerase chain reaction (PCR).

Embodiment 97

The method of embodiment 96, wherein said first fluid includes one ormore reagents selected from the group consisting of a PCR primer, a PCRtemplate, a polymerase, and a PCR reaction buffer.

Embodiment 98

The method according to any one of embodiments 87-97, wherein saiddevice comprise or is integrated with other microfluidic components.

Embodiment 99

The method of embodiment 98, wherein said other microfluidic componentsselected from the group consisting of PDMS channels, wells, valves.

Embodiment 100

The method of embodiment 98, wherein said device includes or isintegrated with a lab-on-a-chip.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic illustration of a droplet merger module 100with deformable lateral membrane valves 106. Fluid containing droplets108 flows in flow direction 112 through a central channel 102. Aplurality of elements 104 form lateral channels 114 that drain fluidfrom between trapped droplets 108 which then merge into a merged droplet116 and leave behind the drained fluid, e.g. as droplets 110. A pair ofpneumatically actuated lateral membrane valves 106 located at the end ofthe plurality of elements (e.g., pillar structure) is used to change thewidth of the center channel to control the number of trapped droplets.When a threshold number of droplets is reached, the hydraulic pressureon the merged droplet becomes bigger than the surface tension to releaseit. No movement of membrane is required, which is the key to enable highspeed merging.

FIG. 2 schematically illustrates pulse laser induced on-demand membranevalve droplet generation. A PDMS thin membrane is used to totallyseparate the pulse laser induced contamination. The induced bubble candeform the membrane into the aqueous channel, break the stable water-oilinterface, and squeeze out a picoliter droplet into the oil channel.

FIG. 3 shows a schematic illustration of an on-demand droplet generationand fusion platform, that integrates a pulse laser induced on-demandmembrane valve droplet generator and a lateral membrane valve controlleddroplet merging module.

FIG. 4 shows snapshots of the droplet generation process.

FIG. 5 shows time-resolved images of an illustrative droplet mergingprocess. Up to six droplets have been experimentally trapped and mergedon this passive but tunable merging module. In this illustratedembodiment, the merged droplet releases automatically when the number ofdroplets is larger than 6.

FIG. 6 schematically illustrates one embodiment of a platform withparallel droplet generators and the downstream droplet merger forgenerating droplets with multiplexed drug/chemical combinations

FIG. 7 schematically illustrates one embodiment of a platformintegrating uFACS and multiple droplet generators for high-speed singlecell encapsulation and single cell analysis.

FIG. 8 illustrates multiple droplet trapping and merging with avertically deformed membrane valve.

FIGS. 9A through 9ZB depict, via simplified cross-sectional views,various stages of a manufacturing technique for producing multi-layerPDMS structures.

DETAILED DESCRIPTION

Two-phase flow (or multi-phase) systems that can manipulate picoliter(μL) volume droplets in closed channels have broad lab on chipapplications. Small droplets allow reduction of reagent consumption,high sensitivity detection, and large scale analysis. Thousands ofdroplets can be easily generated in a simple two-phase flow channel, andtens or hundreds of channels can be structured in parallel to increasethroughput. Droplets can also be programmed to merge, split, and mix athigh speed for rapid screening. Commercial systems could achieve 10million droplet reactions or screening per hour. Applications include avariety of PCR techniques, such as digital PCR, RT-PCR, PCR, single cellanalysis, combinatorial chemical synthesis, and the like.

I. On-Demand Lateral Membrane Valve for Passive Droplet Trapping andMerging

In various embodiments a tunable droplet trapping and merging module isprovided. One embodiment of such a component/module 100 is illustratedin FIG. 1. As illustrated therein the droplet merger component comprisesa central channel 102 comprising a plurality of elements 104 (e.g., anarray of pillar structures) disposed and spaced to create a plurality oflateral passages 114 that drain a carrier fluid out of a fluid streamcomprising droplets 108 of a first fluid contained in the carrier fluid;and a deformable lateral membrane valve 106 disposed to control thewidth of the center channel.

Droplets in, for example, a microchannel 118 move in a downstreamdirection 112. Droplets flowing downstream are trapped in the dropletmerging module. The plurality of elements 104 provides lateral passages114 are used to drain out fluid oil between droplets to merge them. Apair of pneumatically actuated lateral membrane valves 106 located atthe downstream end of the plurality of elements can be used to changethe width of the flow channel. In various embodiments the width of thecentral channel reduces as a function of distance downstream through aplurality of lateral passages. In certain embodiments the width rangesfrom 10 μm to about 1 mm at the upstream end of the component to a widththat ranges about 1 μm to about 900 μm where the downstream width issmaller than the upstream width. In certain embodiments the width of thelateral passages is smaller than the width of the central channel at thesame location and smaller than the average diameter of a droplet in thecentral channel.

In certain embodiments the plurality of elements comprise a micropillararray. In certain embodiments the micropillar array comprises pairs ofpillars that define the central channel. In certain embodiments thevalve is located at or downstream of the last of the plurality ofelements (e.g. downstream of the last (downstream) pair of pillars). Incertain embodiments the pillars are configured to provide aninter-pillar spacing that ranges from about 0.1 μm to about 100 μm. Incertain embodiments the pillars are configured to provide aninter-pillar spacing that ranges from about 0.1 μm to about 10 μm. Incertain embodiments the deformable lateral membrane valve is configuredto form a controllable, variable-sized construction at the downstreamend of said plurality of elements. In certain embodiments the deformablelateral membrane valve is configured to deform vertically.

In various embodiments the deformation of the lateral membrane can betuned by changing the pneumatic pressure though the vias underneath thedeformation chamber. It is noted that in certain embodiments,deformation of the lateral membrane can be regulated by mechanicalactuators. For example, in certain embodiments, piezo-linear actuators,electrostatic actuators, and the like can be used to control deformationof the lateral membrane.

The number of droplets trapped in the merging module can be tuned bydegree of deformation of the lateral membrane valve. When the number oftrapped droplets reaches to its trapping threshold, the fused dropletreleases automatically without the need to mechanically deform themembrane valve. Such passive type droplet merger can have high speedsince there is no need to deform the mechanical membrane. As illustratedin FIG. 5, six droplets can be passively trapped, merged and released.In some cases where more droplets are required to merge (for example,creating a large combinatorial library), the membrane valve, which inthis illustration is deformed vertically can be fully closed to holdmore droplets. Up to 15 droplets have been trapped and merged using thismode, as shown in FIG. 8. It will be noted, however, that the methodsand devices are not limited to trapping six or 15 droplets. Accordinglyin certain embodiments, at least 2, or 3, or 4, or 5, or 6, or 7, or 8,or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or19, or 20, or 21, or 22, or 23, or 24, or 25, or 26, or 27, or 28, or29, or 30 or more droplets are trapped and merged.

In certain embodiments, where the membrane closing process is slow, thethroughput may be lower than about 10 merged droplets/sec.

One illustrative, but non-limiting schematic of an integrated dropletgeneration and merging modules for high speed production of multiplexeddroplets is shown in FIG. 3.

In certain embodiments the devices described herein utilize a thin-layersoft lithography process to produce certain structures (e.g., valvemembranes). The fabrication of thin layers of, e.g., PDMS is enabled bya novel Pt-PDMS thin film process described in copending provisionalapplication No. 61/616,385, filed on Mar. 27, 2012, and in copendingprovisional application entitled “CONTINUOUS WHOLE-CHIP 3-DIMENSIONALDEP CELL SORTER AND RELATED FABRICATION METHOD”, filed on Mar. 15, 2013,both of which are incorporated herein for the PtPDMS thin filmfabrication processes described therein.

In particular one implementation of this ptPDMS fabrication process isdepicted via simplified cross-sectional views in FIGS. 9A through 9ZB.The structure that is being constructed in FIGS. 9A through 9ZB is aportion of a three-dimensional DEP cell sorter, e.g., the featureswithin the DEP separation region of such a cell sorter, however the samefabrication method is readily applied to fabrication of the devicesdescribed herein (e.g., on-demand lateral membrane valve). FIGS. 9Athrough 9ZB are not drawn to scale. In FIGS. 9A through 9P, the Figuresdepict two different manufacturing streams—the steps in the streams maybe largely the same, but the molds used may have different featuresizes. For example, the cross-sections on the left side of each Figuredepict the formation of a PDMS layer that may be used to provide a firstpassage or a second passage, e.g., of a cell sorter, droplet injector,and the like, and the cross-sections on the right side of each Figuremay depict the formation of a PDMS layer that may be used to provide asorting passage of the cell sorter, injection passage of a dropletformer, and the like. FIGS. 9Q through 9ZB depict the assembly of thelayers into an assembled cell sorter (e.g., as described in copendingprovisional application No. 61/616,385, filed on Mar. 27, 2012,).

As illustrated in FIG. 9A, a hard substrate may be prepared for etchingby depositing or providing a photo-patternable or photo-resistivematerial on the substrate. Such a material may be, for example, negativephotoresist SU8 or positive photoresist AZ4620, and the substrate may,for example, be silicon or glass, although other photoresists orphoto-patternable materials may be used as well, as well as othersubstrate materials. As illustrated in FIG. 9B, an etching operation canremove material from the hard substrate to form a master mold.Alternatively, the raised features on the master mold can be formed bydeposition instead of etching. In certain embodiments both etching anddeposition can be used to form features on the master mold. As shown inFIG. 9C, the master mold is coated with a conformal silane surfacetreatment to facilitate later removal of cured PDMS from the molds. InFIG. 9D, uncured PDMS (or other soft lithographic material) may bepoured onto the master mold and cured to form a complementary PDMS mold.In FIG. 9E, the PDMS mold may then be separated from the master mold. InFIG. 9F, the PDMS mold may be coated with a conformal silane surfacetreatment.

As illustrated in FIG. 9G, the PDMS mold may be temporarily set asideand another hard substrate, e.g., silicon or glass, may be prepared bypouring uncured PDMS onto the substrate. In FIG. 9H, the PDMS mold maybe retrieved, and in FIG. 9I, the PDMS mold may be pressed into theuncured PDMS on the substrate and the uncured PDMS may then be cured. InFIG. 9J, the PDMS mold may be removed from the cured PDMS on thesubstrate. The resulting PDMS structure on the substrate may be anexact, or near-exact, duplicate of the master mold and may be referredto herein as a PDMS master mold. It will be recognized that a hardmaster mold or a “soft” (e.g., PDMS) master mold can be used. A hardmaster mold will reduce thin film distortion during the molding process.In standard soft lithography, people use SU-8 mold (a hard master mold)to make PDMS structures.

In FIG. 9K, the PDMS master mold may be coated with a CYTOP™ surfacetreatment to assist in later removal of cast PDMS parts.

Uncured PDMS may be applied to the PDMS master mold (see, e.g., FIG.9L). The steps discussed above with respect to FIGS. 9A through 9L aresimilar, in large part, to existing PDMS layer fabrication processes.

FIG. 9M, however, depicts a step that deviates from existing fabricationtechniques. In existing fabrication techniques, a PDMS stamping, e.g., alarge, flat, featureless base, may be used to compress the uncured PDMSinto the PDMS master mold. In one embodiment of the present fabricationtechnique, however, the PDMS stamping has been modified to include aplate of material within the PDMS that has a much higher modulus thanthe PDMS (e.g., is stiffer than the PDMS). The plate is located suchthat a very thin layer of PDMS exists between the plate and the uncuredPDMS and the PDMS master mold. This thin layer may be, for example, onthe order of 500 microns or less in thickness. In practice, thicknessesof 10 to 30 microns have been found to work well. The plate may beplastic, glass, or other material with a substantially higher modulusthan that of PDMS. In practice, plastic plates have proven to be morerobust than glass plates. Without being bound to a particular theory,the plate may act as an intermediate load spreader within the PDMSstamping to distribute a compression load across the PDMS master moldand the uncured PDMS. The thin layer of PDMS can allow for very smalllocalized deflections that facilitate full contact between the PDMS moldand the stamping while avoiding the creation of large edge ridges thatmay appear when a traditional stamping is used.

In one illustrative, but non-limiting embodiment, the embedded-platestamping shown may be provided by spin-coating the plate with PDMS.However, it was discovered that PDMS exhibits inconsistent curingbehavior when applied in too thin a layer. Indeed, in many thin filmsituations, it was observed that the PDMS does not cure at all andremains in a liquid state. Thus, it was discovered that the PDMS willnot reliably set at thicknesses such as those discussed above, resultingin an unreliable manufacturing technique. It was a surprising discoverythat if the PDMS that forms the thin layer on the stamping is doped witha catalyst (e.g., a platinum catalyst), however, the PDMS will setreliably regardless of thickness. While catalysts have been used toaccelerate cure rate it is believed that such catalysts have not beenpreviously used to reverse a non-cure or inconsistent cure situation, orto prepare PDMS high-temperature processing. Thus, in certainembodiments, the fabrication techniques contemplated herein may includepreparing a stamping (this step is not shown) by coating a substantiallyrigid plate with a thin layer of platinum-doped PDMS. It was discoveredthat providing platinum ions to the uncured PDMS insures consistent,relatively uniform, curing. It was also surprisingly discovered thatwith addition of enough platinum ions, the PDMS cured in a short timeeven at room temperature. In certain embodiments the catalyst isplatinum-divinyltetramethyldisiloxane (C₈H₁₈OPtSi₂).

The stamping may also have a thicker layer of PDMS on the opposite sideof the plate to allow for easy handling or integration with existingequipment, although such a thicker layer is not strictly necessary. Thethin layer of PDMS (or the entire PDMS stamping) may be treated with asilane surface treatment, e.g.,trichloro(1H,1H,2H,2H-perfluorooctyl)silane (also referred to as“PFOCTS”).

In FIG. 9N, the stamping has been compressed against the uncured PDMSand the PDMS master mold and then cured. In FIG. 9O, the cured PDMSlayer is removed from the PDMS master mold by pulling the stamping awayfrom the PDMS master mold. Due to the higher bond strength insilane-treated surfaces as compared with CYTOP-treated surfaces, thePDMS layer will stay bonded to the stamping, allowing for easy transferto other structures.

FIG. 9P depicts the removed PDMS layer bonded to the stamping; the PDMSlayer may be treated with an oxygen plasma to facilitate later bondingwith a glass or PDMS structure. In FIG. 9Q, one of the PDMS layers ispositioned over a prepared glass substrate; the glass substrate may, forexample, be prepared by coating it with an electrically-conductivecoating such as ITO so that it may act as an electrode layer of a DEPcell sorter. In FIG. 9R, the PDMS layer may be directly bonded to theglass substrate as a result of the oxygen plasma treatment of the PDMSlayer. In FIG. 9S, the stamping may be removed—due to the higher bondstrength of the direct bonding via oxygen plasma treatment as comparedwith the bond across the silane-treated surfaces, the PDMS layer mayseparate from the stamping and remain cleanly attached to the glasssubstrate. The PDMS layer placed on the substrate in this casecorresponds to a sublayer of an electrically-insulating layer in a DEPcell sorter having a first or second passage in it.

In FIG. 9T, another PDMS layer (in one embodiment time correspondingwith a sublayer of an electrically-insulating layer in a DEP cell sorterhaving a sorting passage in it), may be positioned over thepreviously-placed PDMS layer using the stamping to which it is attached.This second PDMS layer may also be treated with an oxygen plasma tofacilitate direct bonding to the previously-placed PDMS layer. In FIG.9U, the second PDMS layer may be directly bonded to the first PDMS layerby compressing it into the first PDMS layer with the stamping. In FIG.9V, the stamping may be removed in much the same manner as in FIG. 9S.

In FIG. 9W, a third PDMS layer, in this case similar to the first PDMSlayer, may be positioned over the first and second PDMS layers. Thethird PDMS layer, as with the other PDMS layers, may be treated with anoxygen plasma. In FIG. 9X, the third PDMS layer may be directly bondedto the second PDMS layer to form a three-layer stack of PDMS layers thatare fused into one, essentially contiguous, structure. In FIG. 9Y, thestamping may be removed, leaving the 3-layer PDMS structure behind. InFIG. 9Z, the exposed top of the PDMS structure may be prepared forbonding to another hard substrate, e.g., glass. In FIG. 9ZA, the hardsubstrate may be positioned over the assembled PDMS stack, and in FIG.9ZB, the hard substrate may be bonded to the stack.

In various embodiments the resulting structure provides very cleaninter-layer via features, and is particularly well-suited formicrofluidic devices. The above technique may be modified as needed toomit certain steps, add other steps, and otherwise tailor the techniquefor particular design requirements. For example, it may be possible toform features with stepped cross-sections in the molds, thus reducingthe number of individual layers that must be made and bonded together.While the depicted technique was shown for a 3-layer stack of PDMSlayers, more or less PDMS layers may be manufacturing in this manner andassembled into a PDMS layer stack.

II. Pulse Laser Induced on-Demand Membrane Valve Droplet Generation

FIG. 2 schematically illustrates an embodiment of a pulse laser drivenmembrane valve droplet generator. A thin membrane (e.g., a PDMSmembrane) is used to separate a dye channel for laser excitation fromthe sample channel to prevent potential contamination from pulse laserinduced reactive chemicals.

Each laser pulse can trigger a cavitation bubble to deform the membraneto squeeze out a droplet through the nozzle into the oil phase. Insteadof using continuous phase flow that consumes huge amount of reagents, astatic pressure source can be used to maintain the stable (e.g.,water-oil) interface. This approach dramatically reduces the consumptionof expensive and precious reagents. After a single droplet is ejectedinto the oil phase, the interface can automatically recover to itsoriginal location in a very short time period since the membrane is onlypartially and locally deformed. The droplet generation rate can go up tohundreds of Hz. The volume of droplets produced on certain embodimentsof this platform is around 80 μL as shown in FIG. 4.

Various embodiments of the devices described herein incorporatemicrochannels (microfluidic channels). The terms “microfluidic channel”or “microchannel” are used interchangeably and refer to a channel havingat least one characteristic dimension (e.g., width or diameter) lessthan 1,000 μm, more preferably less than about 900 μm, or less thanabout 800 μm, or less than about 700 μm, or less than about 600 μm, orless than about 500 μm, or less than about 400 μm, or less than about300 μm, or less than about 250 μm, or less than about 200 μm, or lessthan about 150 μm, or less than about 100 μm, or less than about 75 μm,or less than about 50 μm, or less than about 40 μm, or less than about30 μm, or less than about 20 μm.

In certain embodiments the methods and devices described herein mayutilize immiscible fluids. In this context, the term “immiscible” whenused with respect to two fluids indicates that the fluids when mixed insome proportion, do not form a solution. Classic immiscible materialsare water and oil. Immiscible fluids, as used herein also include fluidsthat substantially do not form a solution when combined in someproportion. Commonly the materials are substantially immiscible whenthey do not form a solution if combined in equal proportions. In certainembodiments immiscible fluids include fluids that are not significantlysoluble in one another, fluids that do not mix for a period of time dueto physical properties such as density or viscosity, and fluids that donot mix for periods of time due to laminar flow.

In addition, such fluids are not restricted to liquids but may includeliquids and gases. Thus, for example, where the droplets are to beformed comprising an aqueous solvent (such as water) any number oforganic compounds such as carbon tetrachloride, chloroform, cyclohexane,1,2-dichloroethane, dichloromethane, diethyl ether, dimethyl formamide,ethyl acetate, heptane, hexane, methyl-tert-butyl ether pentane,toluene, 2,2,4-trimethylpentane, and the like are contemplated. Variousmutually insoluble solvent systems are well known to those skilled inthe art (see e.g. Table 1). In another example, droplets of aqueousbuffer containing physiologically normal amounts of solute may beproduced in a dense aqueous buffer containing high concentrations ofsucrose. In yet another example, droplets of an aqueous buffercontaining physiologically normal amounts of solute may be produced in asecond aqueous buffer containing physiologically normal amounts ofsolute where the two buffers are segregated by laminar flow. In stillanother example, droplets of a fluid may be produced in a gas such asnitrogen or air.

TABLE 1 illustrates various solvents that are either miscible orimmiscible in each other. The solvent on left column does not mix withsolvents on right column unless otherwise stated. Solvents ImmiscibilityAcetone can be mixed with any of the solvents listed in the column atleft Acetonitrile cyclohexane, heptane, hexane, pentane,2,2,4-trimethylpentane carbon can be mixed with any of the solventslisted in the column at left except tetrachloride water chloroform canbe mixed with any of the solvents listed in the column at left exceptwater cyclohexane acetonitrile, dimethyl formamide, dimethyl sulfoxide,methanol, water 1,2- can be mixed with any of the solvents listed in thecolumn at left except dichloroethane water dichloromethane can be mixedwith any of the solvents listed in the column at left except waterdiethyl ether dimethyl sulfoxide, water dimethyl cyclohexane, heptane,hexane, pentane, 2,2,4-trimethylpentane, water formamide dimethylcyclohexane, heptane, hexane, pentane, 2,2,4-trimethylpentane, diethylsolfoxide ether 1,4-dioxane can be mixed with any of the solvents listedin the column at left ethanol can be mixed with any of the solventslisted in the column at left ethyl acetate can be mixed with any of thesolvents listed in the column at left except water heptane acetonitrile,dimethyl formamide, dimethyl sulfoxide, methanol, water hexaneacetonitrile, dimethyl formamide, dimethyl sulfoxide, methanol, aceticacid, water methanol cyclohexane, heptane, hexane, pentane,2,2,4-trimethylpentane methyl-tert-butyl can be mixed with any of thesolvents listed in the column at left except ether water pentaneacetonitrile, dimethyl formamide, dimethyl sulfoxide, methanol, water,acetic acid 1-propanol can be mixed with any of the solvents listed inthe column at left 2-propanol can be mixed with any of the solventslisted in the column at left tetrahydrofuran can be mixed with any ofthe solvents listed in the column at left toluene can be mixed with anyof the solvents listed in the column at left except water 2,2,4-acetonitrile, dimethyl formamide, dimethyl sulfoxide, methanol, watertrimethylpentane water carbon tetrachloride, chloroform, cyclohexane,1,2-dichloroethane, dichloromethane, diethyl ether, dimethyl formamide,ethyl acetate, heptane, hexane, methyl-tert-butyl ether, pentane,toluene, 2,2,4- trimethylpentane

In certain embodiments the first fluid and second fluid need not beimmiscible in each other. In such embodiments, injected droplets can bekept separate from each other simply by adjusting flow rates in themicrochannels and rate of bubble formation to form separated bubbles.

In various embodiments the droplets generated by the devices and methodsdescribed herein can contain or encapsulate a wide variety of materials.In some embodiments the droplets may contain test samples, cells,organelles, proteins, nucleic acids, enzymes, PCR or other testingreagents, biochemicals, dyes, or particulates (for example polymericmicrospheres, metallic microparticles, or pigments). In still otherembodiments a droplet may encapsulate one or more previously generateddroplets. In addition, the invention need not be limited to aqueousdroplet systems. For example, such droplet generating methods anddevices may be used in nanoparticle coating, where materials in organicsolvents can be used to deposit layers on or encapsulate nanoparticles.

As noted above, in some embodiments an opening in a fluid channel can beconfigured as a nozzle. The depth, inner diameter, and outer diameter ofsuch a nozzle can be optimized to control droplet size, dropletuniformity, mixing at the fluid interface, or a combination of these.

In certain embodiments the droplet generation and/or droplet mergercomponents described herein may be provided on a substrate that differsfrom the material that comprises the fluid channels. For example, thefluid channels may be fabricated using an elastomeric material that isdisposed upon a rigid surface. Suitable fluid channel materials includebut are not limited to flexible polymers such as PDMS, plastics, andsimilar materials. Fluid channels may also be comprised of nonflexiblematerials such as rigid plastics, glass, silicon, quartz, metals, andsimilar material. Suitable substrates include but are not limited totransparent substrates such as polymers, plastic, glass, quartz, orother dielectric materials. Other suitable substrate materials includebut are not limited to nontransparent materials such as opaque ortranslucent plastics, silicon, metal, ceramic, and similar materials.

The parameters described above and in the Examples (e.g., flow rate(s),laser intensity, laser frequency/wavelength, channel dimensions,port/nozzle dimensions, channel wall stiffness, location of cavitationbubble formation, and the like) can be varied to optimize dropletformation and/or droplet/particle/cell encapsulation for a particulardesired application.

There are a number of formats, materials, and size scales that may beused in the construction of the droplet generating devices describedherein and in microfluidic devices that may incorporate them. In someembodiments the droplet generating devices and the connecting fluidchannels are comprised of PDMS (or other polymers), and fabricated usingsoft lithography. PDMS is an attractive material for a variety ofreasons, including but not limited to low cost, optical transparency,ease of molding, and elastomeric character. PDMS also has desirablechemical characteristics, including compatibility with both conventionalsiloxane chemistries and the requirements of cell culture (e.g. lowtoxicity, gas permeability). In an illustrative soft lithography method,a master mold is prepared to form the fluid channel system. This mastermold may be produced by a micromachining process, a photolithographicprocess, or by any number of methods known to those with skill in theart. Such methods include, but are not limited to, wet etching,electron-beam vacuum deposition, photolithography, plasma enhancedchemical vapor deposition, molecular beam epitaxy, reactive ion etching,and/or chemically assisted ion beam milling (Choudhury (1997) TheHandbook of Microlithography, Micromachining, and Microfabrication, Soc.Photo-Optical Instru. Engineer.; Bard & Faulkner, Fundamentals ofMicrofabrication).

Once prepared the master mold is exposed to a pro-polymer, which is thencured to form a patterned replica in PDMS. The replica is removed fromthe master mold, trimmed, and fluid inlets are added where required. Thepolymer replica may be optionally be treated with a plasma (e.g. an O₂plasma) and bonded to a suitable substrate, such as glass. Treatment ofPDMS with O₂ plasma generates a surface that seals tightly andirreversibly when brought into conformal contact with a suitablesubstrate, and has the advantage of generating fluid channel walls thatare negatively charged when used in conjunction with aqueous solutions.These fixed charges support electrokinetic pumping that may be used tomove fluid through the device. While the above described fabrication ofa droplet generating device using PDMS, it should be recognized thatnumerous other materials can be substituted for or used in conjunctionwith this polymer. Examples include, but are not limited to, polyolefinplastomers, perfluoropolyethylene, polyurethane, polyimides, andcross-linked phenol/formaldehyde polymer resins.

In some embodiments single layer devices are contemplated. In otherembodiments multilayer devices are contemplated. For example, amultilayer network of fluid channels may be designed using a commercialCAD program. This design may be converted into a series oftransparencies that is subsequently used as a photolithographic mask tocreate a master mold. PDMS cast against this master mold yields apolymeric replica containing a multilayer network of fluid channels.This PDMS cast can be treated with a plasma and adhered to a substrateas described above.

As noted above, the methods and devices described herein areparticularly suitable for use in microfluidic devices. In someembodiments therefore the fluid channels are microchannels. Suchmicrochannels have characteristic dimensions ranging from about 100nanometers to 1 micron up to about 500 microns. In various embodimentsthe characteristic dimension ranges from about 1, 5, 10, 15, 20, 25, 35,50 or 100 microns up to about 150, 200, 250, 300, or 400 microns. Insome embodiments the characteristic dimension ranges from about 20, 40,or about 50 microns up to about 100, 125, 150, 175, or 200 microns. Invarious embodiments the wall thickness between adjacent fluid channelsranges from about 0.1 micron to about 50 microns, or about 1 micron toabout 50 microns, more typically from about 5 microns to about 40microns. In certain embodiments the wall thickness between adjacentfluid channels ranges from about 5 microns to about 10, 15, 20, or 25microns.

In various embodiments the depth of a fluid channel ranges from 5, 10,15, 20 microns to about 1 mm, 800 microns, 600 microns, 500 microns, 400microns, 300 microns, 200 microns, 150 microns, 100 microns, 80 microns,70 microns, 60 microns, 50 microns, 40 microns, or about 30 microns. Incertain embodiments the depth of a fluid channel ranges from about 10microns to about 60 microns, more preferably from about 20 microns toabout 40 or 50 microns. In some embodiments the fluid channels can beopen; in other embodiments the fluid channels may be covered.

As noted above, some embodiments a nozzle is present. In certainembodiments where a nozzle is present, the nozzle diameter can rangefrom about 0.1 micron, or about 1 micron up to about 300 microns, 200microns, or about 100 microns. In certain embodiments the nozzlediameter can range from about 5, 10, 15, or 20 microns up to about 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or about 80 microns. In someembodiments the nozzle diameter ranges from about 1, 5, 10, 15, or 20microns to about 25, 35, or 40 microns.

In some embodiments the methods and devices described herein cangenerate droplets at a rate ranging from zero droplets/sec, about 2droplets/sec, about 5 droplets/sec, about 10 droplets/sec, about 20droplets/sec, about 50 droplets/sec, about 100 droplets/sec, about 500droplets/sec, or about 1000 droplets/sec, up to about 1,500droplets/sec, about 2,000 droplets/sec, about 4,000 droplets/sec, about6,000 droplets/sec, about 8,000 droplets/sec, about 10,000 droplets/sec,about 20,000 droplets/sec, about 50,000 droplets/sec, and about 100,000droplets/sec.

In various embodiments the devices and methods described herein cangenerate droplets having a substantially continuous volume. Dropletvolume can be controlled to provide volumes ranging from about 0.1 fL,about 1 fL, about 10 fL, and about 100 fL to about 1 microliter, about500 nL, about 100 nL, about 1 nL, about 500 μL or about 200 pL. Incertain embodiments volume control of the droplet ranges from about 1 pLto about 150 pL, about 200 pL, about 250 pL, or about 300 pL.

As indicate above, the microchannel droplet formation/merger injectiondevices described herein can provide a system integrated with otherprocessing modules on a microfluidic “chip” or in flow throughfabrication systems for microparticle coating, microparticle drugcarrier formulation, and the like. These uses, however, are merelyillustrative and not limiting.

Microfluidic devices can manipulate volumes as small as severalnanoliters. Because the microfluidic reaction volume is close to thesize of single mammalian cells, material loss is minimized insingle-cell mRNA analysis with these devices. The ability to processlive cells inside microfluidic devices provides a great advantage forthe study of single-cell transcriptomes because mRNA is rapidly degradedwith cell death. A highly integrated microfluidic device, having 26parallel 10 nL reactors for the study of gene expression in single humanembryonic stem cells (hESC) has been reported (Zhong et al. (2008) Labon a Chip, 8: 68-74; Zhong et al. (2008) Curr. Med. Chem., 15:2897-2900). In various microfluidic devices all systems for obtainingsingle-cell cDNA including cell capture, mRNA capture/purification, cDNAsynthesis/purification, are performed inside the device. The presentdevices and methods offer effective means of encapsulating and and/orseparating individual cells for, e.g., further processing,

Any of a number of approaches can be used to convey the fluids, ormixtures of droplets, particles, cells, etc. along the channels of thedevices described herein. Such approaches include, but are not limitedto gravity flow, syringe pumps, peristaltic pumps, electrokinetic pumps,bubble-driven pumps, and air pressure driven pumps.

In certain illustrative but non-limiting embodiments two majorapplications of the platforms described herein are contemplated. Theseinclude:

1. Rapid production of a large combinatorial cocktail drug library: A 2Dscanning mirror coupled with a high repetition rate pulse laser cansupport parallel droplet generators deployed on the same microfluidicchip (see, e.g., FIG. 6). The 3D microfluidic fabrication techniquedescribed herein can solve the cross interconnet issues found in 2Dmicrofluidics and enable 3D microchannel routing to support up tohundreds of sample channels on the same chip for producing a largemultiplexed combinatorial library. Producing a large library with 1million different chemical combinations may take less than 3 hours (100combinations/sec).

2. The droplet generation and merging platform described herein can bereadily integrated with our PLACS system described in U. S. PatentPublication No: 2012/0236299 to enable high speed single cellencapsulation and downstream merging of the cell captured droplets withother biochemical reagents such as cell lysing buffers, primers, andother PCR buffers for single cell PCR analysis (FIG. 7). This integratedsystem will enable the first high throughput FACS system that cansimultaneously provide not only optical signatures of single cells butalso the molecular level analysis data such as mRNA expression levels.

While various implementations have been described herein, it should beunderstood that they have been presented by way of example only, and notlimitation. Thus, the breadth and scope of the present disclosure shouldnot be limited by any of the implementations described herein, butshould be defined only in accordance with the following andlater-submitted claims and their equivalents. It is understood that theexamples and embodiments described herein are for illustrative purposesonly and that various modifications or changes in light thereof will besuggested to persons skilled in the art and are to be included withinthe spirit and purview of this application and scope of the appendedclaims. All publications, patents, and patent applications cited hereinare hereby incorporated by reference in their entirety for all purposes.However, where a definition or use of a term in a reference, which isincorporated by reference herein, is inconsistent or contrary to thedefinition of that term provided herein, the definition of that termprovided herein applies and the definition of that term in the referencedoes not apply. The terms “comprises” and “comprising” should beinterpreted as referring to elements, components, or steps in anon-exclusive manner, indicating that the referenced elements,components, or steps may be present, or utilized, or combined with otherelements, components, or steps that are not expressly referenced.

1. A microfluidic droplet merger component, said component comprising: acentral channel comprising a plurality of elements disposed and spacedto create a plurality of lateral passages that drain a carrier fluid outof a fluid stream comprising droplets of a first fluid contained in saidcarrier fluid; and a deformable lateral membrane valve disposed tocontrol the width of said center channel.
 2. The droplet mergercomponent of claim 1, wherein said membrane valve is a pneumaticallyactuated lateral membrane valve.
 3. The droplet merger componentaccording to any one of claims 1-2, wherein the width of said centralchannel reduces as a function of distance downstream through saidplurality of lateral passages.
 4. The droplet merger component accordingto any one of claims 1-3, wherein the where the width of said lateralpassages is smaller than the width of said central channel at the samelocation and smaller than the average diameter of a droplet in thecentral channel.
 5. The droplet merger component according to any one ofclaims 1-4, wherein said plurality of elements comprise a micropillararray.
 6. The droplet merger component according to any one of claims1-5, wherein said valve is located at or downstream of the last of saidplurality of elements.
 7. The droplet merger component of claim 5,wherein said micropillar array comprises pairs of pillars that formlateral channels slanted in a downstream direction.
 8. The dropletmerger component of claim 7, wherein said valve is located at ordownstream of the last (downstream) pairs of pillars.
 9. The dropletmerger component according to any one of claims 1-8, wherein saidpillars are configured to provide an inter-pillar spacing that rangesfrom about 0.1 μm to about 100 μm.
 10. The droplet merger componentaccording to any one of claims 1-8, wherein said pillars are configuredto provide an inter-pillar spacing that ranges from about 0.1 μm toabout 10 μm.
 11. The droplet merger component according to any one ofclaims 1-10, wherein said deformable lateral membrane valve isconfigured to form a controllable, variable-sized construction at thedownstream end of said plurality of elements.
 12. The droplet mergercomponent according to any one of claims 1-11, wherein said deformablelateral membrane valve is configured to deform horizontally.
 13. Thedroplet merger component according to any one of claims 1-11, whereinsaid deformable lateral membrane valve is configured to deformvertically.
 14. The droplet merger component according to any one ofclaims 1-13, wherein said micropillar array is formed from a materialselected from the group consisting of glass, metal, ceramic, mineral,plastic, and polymer.
 15. The droplet merger component according to anyone of claims 1-13, wherein said micropillar array is formed from anelastomeric material.
 16. The droplet merger component of claim 15,wherein said elastomeric material is selected from the group consistingof polydimethylsiloxane (PDMS), polyolefin plastomers (POPs),perfluoropolyethylene (a-PFPE), polyurethane, polyimides, andcross-linked NOVOLAC® (phenol formaldehyde polymer) resin.
 17. Amicrofluidic droplet generator, said generator comprising: a firstmicrofluidic channel containing a first fluid adjacent to a secondmicrofluidic channel containing a second fluid wherein said first fluidis substantially immiscible in second fluid; and a cavitation channel orchamber where the contents of said cavitation channel or chamber isseparated from the contents of said first microfluidic channel by adeformable channel wall or chamber wall, where said cavitation channelor chamber is configured to permit said deformable channel wall orchamber wall to deform when a bubble is formed in said cavitationchannel or chamber, and where said cavitation channel or chamber isdisposed above or below said first microfluidic channel.
 18. The dropletgenerator of claim 17, wherein, said first microfluidic channel is influid communication with said second microfluidic channel via a port ora channel.
 19. The droplet generator according to any one of claims17-18, where a first portion of said first microfluidic channel isdisposed a first distance away from said second microfluidic channel,and a second portion of said first microfluidic channel is disposed asecond distance away from said second microfluidic channel and saidsecond distance is less than said first distance.
 20. The dropletgenerator of claim 19, wherein said first microfluidic channel comprisesa third portion disposed so that said second portion is located betweensaid first portion and said third portion and said third portion of saidmicrofluidic channel is located at a third distance away from saidsecond microfluidic channel and said third distance is greater than saidsecond distance.
 21. The droplet generator according to any one ofclaims 17-20, where the maximum width of said first microfluidic channeland/or said second microfluidic channel ranges from about 0.1 μm toabout 500 μm.
 22. The droplet generator according to any one of claims17-20, where the maximum width of said first microfluidic channel and/orsaid second microfluidic channel ranges from about 50 μm to about 100μm.
 23. The droplet generator according to any one of claims 17-20,where the width of said first microfluidic channel and/or said secondmicrofluidic channel is about 100 μm.
 24. The droplet generatoraccording to any one of claims 17-23, where the maximum depth of saidfirst microfluidic channel and/or said second microfluidic channelranges from about 0.1 μm to about 500 μm.
 25. The droplet generatoraccording to any one of claims 17-23, where the maximum depth of saidfirst microfluidic channel and/or said second microfluidic channelranges from about 40 μm to about 80 μm.
 26. The droplet generatoraccording to any one of claims 17-23, where the typical depth of saidfirst microfluidic channel and/or said second microfluidic channel isabout 50 μm.
 27. The droplet generator according to any one of claims17-26, wherein the typical depth of said cavitation channel or chamberranges from about 100 μm to about 150 μm.
 28. The droplet generatoraccording to any one of claims 17-27, wherein said droplet generator isconfigured to generate droplets having a volume ranging from about 1atto L to about 1 μL.
 29. The droplet generator of claim 28, whereinsaid droplet generator is configured to generate droplets having avolume ranging from about 1 μL to about 150 μL.
 30. The dropletgenerator according to any one of claims 17-28, wherein said cavitationchannel or chamber is a cavitation channel.
 31. The droplet generator ofclaim 30, wherein said cavitation channel provides permits the contentsof said channel to flow and thereby aid dissipation of a bubble formedtherein.
 32. The droplet generator according to any one of claims 17-31,wherein said cavitation channel or chamber is disposed above said firstmicrofluidic channel.
 33. The droplet generator according to any one ofclaims 17-31, wherein said cavitation channel or chamber is disposedbelow said first microfluidic channel.
 34. The droplet generatoraccording to any one of claims 17-33, wherein said cavitation channel orchamber contains a dye.
 35. The droplet generator according to any oneof claims 17-33, wherein said cavitation channel or chamber containslight-absorbing nanoparticle and/or microparticles.
 36. The dropletgenerator according to any one of claims 17-35, wherein said firstmicrofluidic channel is configured to provide said first fluid under asubstantially static pressure to create a stable interface between saidfirst fluid and said second fluid.
 37. The droplet generator accordingto any one of claims 17-36, wherein said first fluid comprises anaqueous fluid.
 38. The droplet generator according to any one of claims17-37, wherein said second fluid comprises an oil or an organic solvent.39. The droplet generator of claim 38, wherein said second fluidcomprises a solvent selected from the group consisting of carbontetrachloride, chloroform, cyclohexane, 1,2-dichloroethane,dichloromethane, diethyl ether, dimethyl formamide, ethyl acetate,heptane, hexane, methyl-tert-butyl ether, pentane, toluene, and2,2,4-trimethylpentane.
 40. The droplet generator of claim 38, whereinsaid second fluid comprises an oil.
 41. The droplet generator accordingto any one of claims 17-40, wherein said port or channel comprises anozzle.
 42. The droplet generator according to any one of claims 17-41,wherein said first and/or second microfluidic channel is formed from amaterial selected from the group consisting of glass, metal, ceramic,mineral, plastic, and polymer.
 43. The droplet generator according toany one of claims 17-42, wherein said first and/or second microfluidicchannel is formed from an elastomeric material.
 44. The dropletgenerator of claim 43, wherein said elastomeric material is selectedfrom the group consisting of polydimethylsiloxane (PDMS), polyolefinplastomers (POPs), perfluoropolyethylene (a-PFPE), polyurethane,polyimides, and cross-linked NOVOLAC® (phenol formaldehyde polymer)resin.
 45. The droplet generator according to any one of claims 17-44,wherein said generator can provide on-demand droplet generation at aspeed of greater than about 1,000, more preferably greater than about2,000 droplets/sec, more preferably greater than about 4,000droplets/sec, more preferably greater than about 6,000 droplets/sec, ormore preferably greater than about 8,000 droplets/sec.
 46. The dropletgenerator according to any one of claims 17-44, wherein said device canprovide on-demand droplet generation at a speed ranging from zerodroplets/sec, 1 droplets/sec, 2 droplets/sec, about 5 droplets/sec,about 10 droplets/sec, about 20 droplets/sec, about 50 droplets/sec,about 100 droplets/sec, about 500 droplets/sec, or about 1000droplets/sec, up to about 1,500 droplets/sec, about 2,000 droplets/sec,about 4,000 droplets/sec, about 6,000 droplets/sec, about 8,000droplets/sec, about 10,000 droplets/sec, about 20,000 droplets/sec,about 50,000 droplets/sec, or about 100,000 droplets/sec.
 47. Thedroplet generator according to any one of claims 17-44, wherein saiddevice can provide on-demand droplet generation at a speed of greaterthan about 1,000, more preferably greater than about 10,000, morepreferably greater than about 20,000 droplets/sec, more preferablygreater than about 40,000, more preferably greater than about 50,000droplets/sec, more preferably greater than about 80,000, or morepreferably greater than about 100,000 droplets/sec.
 48. The dropletgenerator according to any one of claims 17-47, wherein said generatoris present in a system comprising an energy source configured to form abubble in said cavitation channel or chamber.
 49. The droplet generatorof claim 48, wherein said energy source comprises an optical energysource or microwave emitter.
 50. The droplet generator of claim 48,wherein said energy source comprises a laser.
 51. The droplet generatorof claim 50, wherein said energy source comprises a pulse laser.
 52. Thedroplet generator according to any one of claims 17-51, wherein saidgenerator is disposed on a substrate comprising a material selected fromthe group consisting of a polymer, a plastic, a glass, quartz, adielectric material, a semiconductor, silicon, germanium, ceramic, and ametal or metal alloy.
 53. The droplet generator according to any one ofclaims 17-52, wherein said generator is integrated with othermicrofluidic components.
 54. The droplet generator of claim 53, whereinsaid other microfluidic components selected from the group consisting ofPDMS channels, wells, valves.
 55. The droplet generator of claim 53,wherein said generator is a component of a lab-on-a-chip.
 56. Thedroplet generator according to any one of claims 17-55, wherein saidfirst fluid comprises one or more reagents for polymerase chain reaction(PCR).
 57. The droplet generator of claim 56, wherein said first fluidcomprises one or more reagents selected from the group consisting of aPCR primer, a PCR template, a polymerase, and a PCR reaction buffer. 58.A device for the manipulation of microfluidic droplets, said devicecomprising a substrate carrying or comprising: one or more dropletmerger components according to any one of claims 1-16; and optionallyone or more droplet generators according to any one of claims 17-57. 59.The device of claim 58, wherein said device further comprises acontroller that controls the amount and timing of constriction of saidmembrane valve.
 60. The device according to any one of claims 58-59,wherein said device comprises one or more droplet generators accordingto any one of claims 17-57.
 61. The device according to any one ofclaims 58-60, wherein said device comprises at least two dropletgenerators.
 62. The device of claim 61, wherein said device comprises atleast four droplet generators.
 63. The device according to any one ofclaims 61-62, wherein a plurality of droplet generators are configuredto share a common second microfluidic channel and to inject dropletsinto said common second microfluidic channel.
 64. The device of claim63, wherein a droplet merger component is disposed to receive and mergedroplets from said common second microfluidic channel.
 65. A system forthe generation of droplets and/or the encapsulation of particles orcells said, said system comprising a droplet generator according to anyone of claims 17-57 and an excitation source for forming gas bubbles ina fluid.
 66. The system of claim 65, wherein said excitation sourcecomprises an optical energy source.
 67. The system of claim 66, whereinsaid excitation source comprises a non-coherent optical energy source.68. The system of claim 66, wherein said excitation source comprises alaser.
 69. The system according to any one of claims 66-68, wherein saidsystem comprises an objective lens configured to focus optical energyinto said cavitation channel or chamber.
 70. The system of claim 69,wherein said system comprises a half-wave plate.
 71. The systemaccording to any one of claims 69-70, wherein said system comprises apolarizer.
 72. The system of claim 71, wherein said polarizer comprisesa polarizing beam splitter cube.
 73. The system according to any one ofclaims 66-72, wherein said system comprises a controller that adjusts atleast one of the timing of occurrence of light pulses emitted by theoptical energy source, the frequency of occurrence of pulses emitted bythe optical energy source, the wavelength of pulses emitted by theoptical energy source, the energy of pulses emitted by the opticalenergy source, and the aiming or location of pulses emitted by theoptical energy source.
 74. The system according to any one of claims65-73, wherein said system further comprises components for detectingparticles, droplets, or cells in said system.
 75. The system of claim74, wherein said components comprise an optical detection system, anelectrical detection system, a magnetic detection system or an acousticwave detection system.
 76. The system of claim 74, wherein saidcomponents comprise an optical detection system for detecting ascattering, a fluorescence, or a ramen spectroscopy signal.
 77. A methodof combining droplets in a microfluidic system, said method comprisingproviding a plurality of droplets flowing through a microfluidic channelinto the central channel(s) of one or more droplet merger componentsaccording to any one of claims 1-16 causing the merger of a plurality ofdroplets.
 78. The method of claim 77, further comprising varying theconstriction created by said lateral membrane valve to control thetiming of droplet merger and/or the number of merged droplets.
 79. Themethod of claim 78, wherein said varying the constriction comprisesoperating a controller that pneumatically actuates said lateral membranevalve(s).
 80. A method for generating droplets said method comprising:applying an energy source to a droplet generator according to any one ofclaims 17-57, where said energy source forms bubbles in said cavitationchannel or chamber to deform said deformable channel wall or chamberwall and to inject a droplet of said first fluid into said second fluidin said second microfluidic channel.
 81. The method of claim 80, whereinsaid utilizing an energy source comprises utilizing a laser to excitecavitation bubbles in said cavitation channel or chamber.
 82. The methodof claim 81, wherein said method comprises using a controller thatadjusts at least one of the timing of occurrence of pulses emitted by apulsed laser, the frequency of occurrence of pulses emitted by thepulsed laser, the wavelength of pulses emitted by the pulse laser, theenergy of pulses emitted by the pulse laser, and the aiming or locationof pulses emitted by the pulse laser.
 83. The method according to anyone of claims 80-82, further comprising generating a plurality ofseparate and additional cavitation bubbles at a frequency of at least1000 Hz.
 84. The method of claim 83, wherein said method is repeated ata frequency of 1 kHz or greater.
 85. The method according to any one ofclaims 80-84, wherein said first fluid comprises one or more reagentsfor polymerase chain reaction (PCR).
 86. The droplet generator of claim85, wherein said first fluid comprises one or more reagents selectedfrom the group consisting of a PCR primer, a PCR template, a polymerase,and a PCR reaction buffer.
 87. A method of generating and combiningdroplets, said method comprising: providing a device comprising one ormore droplet generators droplet generator according to any one of claims17-57 and one or more droplet merger component(s) according to any oneof claims 1-16, wherein at least one of said one or more droplet mergercomponent(s) is disposed to receive droplets generated by at least oneof said one or more droplet generators; applying an energy source acavitation channel or chamber of one or more of said one or more dropletgenerator(s), where said energy source forms bubbles in said cavitationchannel or chamber to deform said deformable channel wall or chamberwall and to inject a droplet of said first fluid into said second fluidin said second microfluidic channel; receiving a plurality of dropletsgenerated by said one or more droplet generator(s) in at least one ofsaid one or more droplet merger components where said droplets merger toform a combined droplet fluid.
 88. The method of claim 87, wherein saiddevice comprises a plurality of droplet generators.
 89. The method ofclaim 87, wherein said device comprises at least three dropletgenerators.
 90. The method according to any one of claims 87-89, whereinsaid device comprises a plurality of droplet merger components.
 91. Themethod of claim 90, wherein said device comprises at least three dropletmerger components.
 92. The method according to any one of claims 87-91,wherein said utilizing an energy source comprises utilizing a laser toexcite cavitation bubbles in said cavitation channel or chamber.
 93. Themethod of claim 92, wherein said method comprises using a controllerthat adjusts at least one of the timing of occurrence of pulses emittedby a pulsed laser, the frequency of occurrence of pulses emitted by thepulsed laser, the wavelength of pulses emitted by the pulse laser, theenergy of pulses emitted by the pulse laser, and the aiming or locationof pulses emitted by the pulse laser.
 94. The method according to anyone of claims 87-93, further comprising generating a plurality ofseparate and additional cavitation bubbles at a frequency of at least1000 Hz.
 95. The method of claim 94, wherein said method is repeated ata frequency of 1.2 kHz or greater.
 96. The method according to any oneof claims 87-95, wherein said first fluid comprises one or more reagentsfor polymerase chain reaction (PCR).
 97. The method of claim 96, whereinsaid first fluid comprises one or more reagents selected from the groupconsisting of a PCR primer, a PCR template, a polymerase, and a PCRreaction buffer.
 98. The method according to any one of claims 87-97,wherein said device comprise or is integrated with other microfluidiccomponents.
 99. The method of claim 98, wherein said other microfluidiccomponents selected from the group consisting of PDMS channels, wells,valves.
 100. The method of claim 98, wherein said device comprises or isintegrated with a lab-on-a-chip.